Studies On The Molecular Mechanism Of PPARG Different Splicers Regulating Fatty Acid Synthesis In Buffalo Mammary Epithelial Cells

Buffalo milk is rich in nutrients and has anti-cancer and cardiovascular protective effects.The main nutrient in buffalo milk is butterfat and is rich in beneficial fatty acids such as CLA and EPA.There are two sources of fatty acids in buffalo mammary tissue,these include long-chain fatty acids taken directly from the blood,and another source is short-and medium-chain fatty acids,which are resynthesized by breast cells.After physiological changes such as activation and desaturation,these fatty acids form triglycerides with glycerol,and then gather with a small amount of cholesterol esters to form newborn lipid droplets.The product is coated with a rough endoplasmic reticulin monolayer and then secreted into the cytoplasm to mature.When mature fat droplets fuse together,their volume will increase,and they will be wrapped by cell membrane at the top of the cell and secreted to the outside of the cell,to complete the secretion of milk fat.It has been shown that peroxisome proliferator-activated receptor gamma(PPAR)receptor gamma(PPARG),as a member of the nuclear receptor superfamily,it is involved in regulating various biological processes,such as adipocyte differentiation and lipid metabolism,but how it regulates fatty acid synthesis is not well understood.In this study,we analyzed the differences in fatty acid species and contents in buffalo milk during different lactation periods,and investigated the expression of PPARG gene in buffalo during different lactation stages to verify its influence on fatty acid synthesis.We systematically investigated the effects and molecular mechanisms of PPARGX17 and PPARG-X21 on fatty acid synthesis.The specific findings were as follows:(1)The fat content of buffalo milk in middle and late lactation was high,and docosahexaenoic acid(DHA)was only present in early and middle lactation.The gene expressions of PPARG-X17 and PPARG-X21 were detected in the milk of buffalo in early lactation,middle lactation and late lactation,and the expression of PPARG-X21 in early and middle lactation was significantly higher than that in late lactation.(2)The sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are non-secretory hydrophilic proteins acting in the nucleus,predicting potential phosphorylation and hematoxylation protein modification sites could regulate the activity and function of PPARG.Amino acid sequence predictions showed significant differences in secondary and tertiary structure,and only the protein encoded by the X21 has a PPAR ligand binding domain(NR＿LBD＿PPAR),which is the main location where PPARG exerts transcriptional activity.This structural difference may also determine the functional differences between the two in regulating fatty acid synthesis.(3)After RNA interference/overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells,both were found to differentially inhibit/promote fatty acid synthesis in buffalo mammary epithelial cells,probably mainly by affecting ACACA(fatty acid activation-associated),CD36,ACSL1(fatty acid uptake and transport related genes),GPAT,AGPAT6,DGAT1(triglyceride synthesis related genes)to regulate fatty acid synthesis.The results of this study suggest that PPARG-X17 and PPARG-X21 are continuously expressed during lactation and may play a key regulatory role in the fatty acid synthesis in buffalo milk.The present study resolved the molecular mechanism of fatty acid synthesis in buffalo mammary epithelial cells,which not only provides an important reference for improving milk fat rate,but also provides theoretical support for breed improvement of dairy buffalo in China.